Stimulus-response coupling mediated by changes in intracellular Ca2+ involves the participation of a family of structurally related Ca2+-binding proteins, which act as sensors and modulators of Ca2+ transients. These proteins undergo a conformational change upon binding Ca2+ that enables them to activate target enzymes. Calmodulin is a unique member of this protein family because of its ability to activate a large number of enzymes, in particular protein kinases and phosphatases. Our goal is to further our understanding of the mechanism of action of calmodulin using as a model system the calmodulin stimulation of calcineurin, the only protein phosphatase under the direct control of calmodulin. The identification of calcineurin as the target of the immunosuppressive drugs Cyclosporin A and FK506 by S. Schreiber and his colleagues revealed the key role of this enzyme in the Ca2+-dependent steps of T cell activation. Over the past several years the use of these drugs, which are specific inhibitors of calcineurin, has helped to identify the important roles of calcineurin in the Ca2+/calmodulin regulation of cellular processes as diverse as cell growth and differentiation, programmed cell death, embryogenesis, ion homeostasis, neurotasmitter release, and establishment of long memory. Alteration of calcineurin activity has recently been implicated in an ever increasing number of diseases. In collaboration with Jay Zweier at Johns Hopkins University, we previously showed that calcineurin is an (Fe2+-Zn2+) enzyme which is inactivated by oxidation of Fe2+ to Fe3+. The inactivation of the enzyme is dependent on Ca2+/calmodulin binding to calcineurin and exposure of the active site metal ions. This calmodulin-dependent inactivation, prevented by superoxide dismutase and reversed by ascorbate, provides a mechanism for the temporal regulation of calcineurin in response to Ca2+ transients and the coupling of protein phosphorylation and oxidative stress. During the past year we have devoted our efforts to the elucidation of the role of Ca2+ binding to calcineurin B in the activation of calcineurin. In collaboration with Jill Trewhella (Los Alamos National Laboratory) we have used site directed mutagenesis, flow dialysis, and Fourier transformed infrared (FTIR) spectroscopy to characterize the Ca2+-binding properties of calcineurin B. Unlike calmodulin, and like troponin C, calcineurin B has two high affinity Ca2+/Mg2+-binding sites located in the C-terminal lobe. All four sites communicate with each other and mutation in site 2 (E73/N) leaves the protein with only two titratable sites whose affinities are significantly decreased (Biochemistry, in press). The loss of two Ca2+ sites may be responsible for the inability of one of our summer students, Aram Lee, to reconstitute a fully active calcineurin with this mutant. We are presently investigating the mechanism of this negative effect of the site 2 mutation on Ca2+ binding to the remaining sites with Sergei Ruvinov and Shipeng Li. Like troponin C, in the presence of Ca2+ calcineurin B exists as a monomer-dimer equilibrium with a dissociation constant of 2-4 x 10 -4M. Preliminary evidence suggests that mutation in site 2 dramatically decreases this dissociation constant measured by sedimentation equilibrium. We are presently analyzing the Ca2+ dependence of the dimerization process to further our understanding of the mechanism of communication between the four sites of calcineurin B. Limited proteolysis was used by Seun-Ah Yang to identify the conformational change accompanying Ca2+ binding to the low affinity sites of calcineurin B allowing calmodulin activation and interaction of calcineurin with the immunosuppressive drugs and the transcription factor NF-AT. In the presence of Ca2+ the calmodulin-binding domain of calcineurin A and the inhibitory domain are readily cleaved by proteases. In the absence of Ca2+ (2 mM EGTA) these domains are completely protected against proteolytic attack. Under these conditions the high affinity sites of calcineurin B are still occupied and calcineurin B remains bound to calcineurin A but the low affinity activation sites are depleted of Ca2+. Thus, occupancy of the low affinity sites induces a large conformational change of the regulatory domain of calcineurin A affecting both the calmodulin and inhibitory domains. On the basis of these observations, a mechanism explaining the dependence on Ca2+ binding to calcineurin B for calmodulin activation and for the previously reported 10-20-fold increase in affinity of calcineurin for Ca2+ upon removal of the regulatory domain was presented in a paper published in Biochemistry. An expression system developed by Hao Ren in our laboratory is now being used to obtain large amounts of the b-isoform of calcineurin suitable for the crystallization of calcineurin with the low affinity sites of calcineurin B depleted in order to confirm the structure of this enzyme predicted on the basis of the limited proteolysis experiments. The protein will also be used to try to crystallize calcineurin complexed with calmodulin. These crystallization projects will be done in collaboration with Di Xia (Laboratory of Cell Biology, NCI). The elucidation of the structure of this complex is needed to understand how calmodulin mutants which bind calcineurin with the same affinity as wild type calmodulin, activates its phosphatase activity only partially. With the help of Shipeng Li we have continued our collaborative studies with Ad Bax of calmodulin structure. Calmodulin mutants with an E/N substitution in the last residue of the two N-terminal Ca2+ binding loops were used to demonstrate a Ca2+ induced disruption of the central helix upon occupancy of the two C-terminal sites (manuscript in preparation). This protein was also used by Ad Bax to illustrate the use of heteronuclear dipolar couplings to refine the low resolution structures of proteins predicted on the basis of sequence homology with protein of known structure (this work was published in the J. Biomol. NMR). This method, applied to the characterization of the solution structure of Ca2+-calmodulin, revealed differences between the crystal and solution structures that may be critical to understand the ability of calmodulin to recognize multiple targets (in press, Nat. Struct. Biol., in press).